Use of bronopol for the treatment of diseases in fish

ABSTRACT

The invention relates to the use of bronopol (bromo-2-nitropropane-1,3-diol), in the treatment of various diseases of aquatic organisms, particularly salmonid fish and their eggs. The diseases which can be treated include thgose caused by fungal infections (such as Saprolegnia parasitica); protozoan infections, whether flagellate (such as Ichthyobodo necatrix) or ciliate (such as Icthyophthirius multifiliis); bacteria (such as Flavobacterium branchiophilum); and myxobacteria (such as Cytophaga psychrophila). Also disclosed is a method of disinfecting fish tanks and/or equipment, using a solution of bronopol.

CONTINUING DATA

This application is a 371 of PCT/GB97/02042 filed on Jul. 29, 1997.

This invention relates Go the treatment of fungal infections (especiallySaprolegnia parasitica) of fish, particularly, but not exclusively,trout, salmon, salmonids in general, and their eggs. The invention alsoprovides treatments of other fish diseases such as bacterial infections(for example bacterial gill disease--Flavobacterium branchiophilum andCytophaga psychrophila), protozoan infections such as ciliates (forexample Ichthyophthirius multifiliis) and flagellates (for exampleIchthyobodo necatrix).

Saprolegnia parasitica is a rapidly spreading and fatal fungal parasiteaffecting both fish and fish eggs. It is conventionally treated withmalachite green (diamino-triphenylmethane), but though this treatment ishighly effective, the use of malachite green carries with it a number ofpotential problems: the compound has been suggested as a possiblecarcinogen and teratogen, though these effects are as yet unproven;being a strong dye, it tends to discolour water, and can under certainconditions cause staining of fish which have been treated; it has arelatively long withholding period, so that significant residues can bepresent in treated fish when they are harvested and sold forconsumption; and the compound is not licensed as a veterinarymedicament, and is actually banned from use in U.S. Federal hatcheries.The article by D. J. Alderman in Journal of Fish Diseases 8 (1985)289-298 gives a review of the use of malachite green in treating fishdiseases, and discusses some of the problems associated therewith.

One currently available alternative to malachite green is formalin,which is the substance which is now used in US Federal hatcheries.However, due to the irritancy of this substance, it can only be usedunder strictly controlled conditions. There is therefore a need for animproved treatment for fungal and other diseases of fish, particularlyone which combines high efficacy with low toxicity. Many attempts toidentify new treatments have been made in the past and the results ofthe screening of 40 potential alternative substances are set out in thearticle by D. J. Alderman appearing at Journal of Fish Diseases 5 (1982)113-123, which also sets down standard protocols for the testing ofcandidate treatments. However, despite this and other work (such as thatdescribed in the article by T. A. Bailey appearing at Aquaculture 38(1984) 97-104), the applicants are not aware of any suitable alternativetreatments to malachite green and formalin having reached themarketplace.

It has now been found that bronopol (2-bromo-2-nitropropane-1,3-diol)has good activity at relatively low concentrations against Saprolegniaparasitica, and is safe to use. Bronopol is a known compound, and isused in concentrations of between 0.01 and 0.2% as an antimicrobialpreservative and antiseptic in topical pharmaceutical formulations,cosmetics, and toiletries. It is stated however in "Handbook ofPharmaceutical Excipients" published by The Pharmaceutical Press (1994)that one of bronopol's major disadvantages is its relatively pooractivity against yeasts and moulds. It is therefore surprising thatbronopol is effective at relatively low concentrations againstSaprolegnia.

As will be described in more detail below, bronopol has been shown to beeffective against Saprolegnia parasiticainfection in salmon, trout andtrout eggs. It is envisaged however that the treatment will be effectiveagainst the same infection in other salmonid species, and indeed inother fish (for example ornamental or pet fish), fish eggs and otheraquatic creatures (such as shrimps or prawns) in general. It is expectedthat there will also be efficacy against other fungal infections.Furthermore, as bronopol has been shown in the trials described below toprevent or slow the spread of infection, it may also be used as aprophylactic.

Bronopol is a commercial product available from a number of sources. Itmay be manufactured by reacting nitromethane with paraformaldehyde in analkaline environment, followed by bromination. It is also known thatsome compounds (such as 5-bromo-5-nitro-1,3-dioxane) break down torelease bronopol, and the invention therefore also extends to the use ofsuch compounds in the place of bronopol.

Thus, the invention provides, in one aspect, the use of bronopol, or acompound releasing bronopol in use, in the manufacture of a medicamentfor the treatment or prophylaxis of fungal infections in fish, fish eggsor other aquatic organisms. More specifically, the invention relates tothe use of bronopol in the treatment and/or prophylaxis of Saprolegniaparasitica. One particular use of the invention will be in the treatmentof salmonid fish and their eggs, particularly trout and salmon.

The invention also extends to a method of treating fish, fish eggs orother aquatic organisms suffering from fungal infection by administeringbronopol, or a compound releasing bronopol in use, in a treatment bathin a pharmaceutically effective amount. Preferably the method includesthe step of halting water flow through the bath while the treatment isadministered, preferably for a period of not less than 30 minutes.

Preferably, the concentration of bronopol in the treatment bath is inthe range of 1 mg.l⁻¹ (ppm) to 1000 mg.l⁻¹ more preferably 5 mg.l⁻¹ to200 mg.l⁻¹, and ideally 10 mg.l⁻¹ to 100 mg.l⁻¹. Bronopol is a solidcrystalline substance, and may conveniently be prepared as a solution ina polar solvent, such as water or Dowanol (dipropylene glycolmonoethylether).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 are graphical representations of the results of testingconducted to determine the in vivo efficacy of bronopol againstSaprolegnia parasitica infection in trout eggs.

FIG. 4 is a graphical representation of the results of testing conductedto determine the in vivo efficacy of bronopol against Saprolegniaparasitica infection in salmon.

FIG. 5 is a graphical representation of the results of testing conductedto determine the in vitro efficacy of bronopol against Cytophagapsychrophila.

FIGS. 6-8 are graphical representations of the results of testingconducted to determine the in vivo efficacy of bronopol againstFlavobacterium branchophilum infection in rainbow trout.

Trial 1 (below) was conducted in mildly alkaline conditions(approximately pH 7.4 to 7.5) and it is possible that these conditionsare favourable for the anti-fungal activity demonstrated by bronopol.

During the trials, bronopol was also tested against various other fishdiseases, and was found to be effective against Ichthyobodo necatrix,which is an ectoparasite flagellate protozoan, Flavobacteriumbranchiophilum (the causative organism of bacterial gill disease), andCytophaga psychrophila, which is a myxobacterium and the causativeorganism of Rainbow Trout Syndrome, Coldwater disease, Saddleback andPeduncle disease in salmonid fish. The activity against Cytophagaindicates possible efficacy against myxobacteria in general, and theactivity against Ichthyobodo suggests efficacy against flagellates ingeneral, as well as ciliate protozoans such as Ichthyophthiriusmultifiliis.

Thus, in a further aspect the invention extends to the use of bronopol,or a compound releasing bronopol in use, in the manufacture of amedicament for the treatment or prophylaxis of a protozoan infection,such as a flagellate (for example Ichthyobodo necatrix) or a ciliate(such as Ichthyophthirius multifiliis) in fish or other aquaticorganisms, particularly salmonid fish, and especially trout.

In an alternative aspect, the invention extends to the use of bronopol,or a compound releasing bronopol in use, in the manufacture of amedicament for the treatment or prophylaxis of bacterial gill disease(for example Flavobacterium branchiophilum) in fish or other aquaticorganisms, particularly salmonid fish, and especially trout.

The invention also encompasses a method of treating such protozoan orflavobacterium infections in fish or other aquatic organisms byadministering bronopol, or a compound releasing bronopol in use, in atreatment bath in a pharmaceutically effective amount. Preferably, thebronopol is administered in a concentration of between 1 and 500 mg.l⁻¹,more preferably between 10 and 100 mg.l⁻¹.

The main practical use of the activity against Cytophaga is likely to bein the disinfection of tanks and equipment and the invention thereforeencompasses a method of disinfecting fish tanks and/or instruments foruse in the husbandry of fish, the method comprising the step of exposingthe tank/equipment to a solution containing bronopol, or a compoundreleasing bronopol in use. Preferably, the bronopol is present in aconcentration of between approximately 1 and 2000 mg.l⁻¹, morepreferably 5 to 1000 mg.l⁻¹. The exposure time is preferably between 2and 40 mins.

It is possible that bronopol may also be effective in treating fishsuffering from myxobacterial infection, and the invention therefore alsoextends to the use of bronopol, or a compound releasing bronopol in use,in the manufacture of a medicament for the treatment or prophylaxis ofmyxobacterial infections (particularly Cytophaga psychrophila) of fishor other aquatic organisms, particularly salmonid fish, and especiallytrout.

The invention is hereinafter described in more detail by way of exampleonly, with reference to the following experimental trials:

Trial 1 In Vitro Efficacy of Bronopol Against Saprolegnia parasitica

This trial was carried out using the procedures described in Journal ofFish Diseases (1982) 5, 113-123, cited above.

Preparation of Inocula

A culture of Saprolegnia parasitica was maintained at 16° C. on riverwater, glucose, yeast extract agar (RGY), consisting of yeast extract (1g), D(+)glucose (5 g), and agar (12 g) in 1 l river water. Plates of RGYwere seeded with the test organism and incubated at 25° C. until growthjust covered the full diameter of the dish (approximately 72 hours).Discs were cut from the outer 10 mm of the culture, using a 4-mmdiameter punch (adapted from a gel chromatography well punch bywelding-on a handle) and then used as standard inocula for testing.

Test 1A

Method

Bronopol was tested for activity against the Saprolegnia parasiticacultures at different concentrations ranging from 50mg.l⁻¹ to 100mg.l⁻¹, using Protocol II of the aforementioned J. Fish Diseasesarticle, as follows. Polycarbonate filter membranes of 0.2-μm porosityand 25-mm diameter (Nuclepore; Sterlin) were sterilized by autoclavingand then placed on the surface of RGY plates (7 per 90-mm petri-dish). Astandard 4-mm disc inoculum was then placed, inverted, at the centre ofthe filter. The dishes containing the filters were then incubated untilthe resulting mycelial mat had almost reached the edge of the filters.The original inocula were then clipped off (as far as practical) usinghot forceps tips to avoid disturbing the loosely adherent mycelial mat.The mycelial mats together with their supporting filters were lifted offthe agar surface on the filters, transferred to empty, sterile,petri-dishes and completely submerged in the bronopol solution atselected concentrations. At the end of the 1 hour exposure period thebronopol solution was removed by aspiration and replaced by two washesof sterile river water (for 5 and 30 min, respectively) before themycelial mat and filter were transferred, filter uppermost, to thesurface of a fresh RGY plate. After incubation at 16° C. for 24 h, anynew growth beyond the edge of the filter was measured at four pointsaround the filter, at 90° intervals.

The experiment was performed using six separate culture samples at eachconcentration of bronopol, and was repeated using malachite green inplace of bronopol. A negative control test using no active agent wasalso performed. The results are set out in the table below:

                  TABLE 1A                                                        ______________________________________                                        Radial growth in mm at 24 h after                                             exposure to bath of test concentration                                        Conc  Time   BRONOPOL        MALACHITE GREEN                                  mg · l.sup.-1                                                              (min)  1     2   3   4   5   6   1   2   3   4                                                     5   6                                              ______________________________________                                        1000  10     0     0   0   0   0   0   0   0   0   0                                                     0   0                                                                         500 10 0 0 0 0 0 0 0 0 0 0 0 0                                                100 10 3 3 2 2 1 2 0 0 0 0 0 0                                                 50 10 3 3 3 3 2 2 0 0 0 0 0 0                                                 50  5 5 2 2 3 3 4 0 0 0 0 0 0                                                 10 10 6 7 7 6 7 6 0 0 0 0 0 0                                                 10  5 7 7 7 6 8 8 0 0 0 0 0 0                                                Control                                                                           10 7 7 7 6 7 7                                 ______________________________________                                    

Discussion

In this test naked fungal hyphae are exposed to bronopol, which is shownto have an inhibitory effect as low as 50 ppm for 5 minutes againstsubsequent vegetative growth. Ten minutes' exposure at 500 ppm preventsall subsequent vegetative growth with this test method.

Test 1B

Method

In this test, bronopol was tested for efficacy against Saprolegniaparasitica using the more demanding Protocol III of the aforementionedJ. Fish Diseases article, the method being as follows:

Four disc inocula were placed in each compartment of a sterile replidish(25 compartments in five rows; Sterilin). Five different testconcentrations applied for five different exposure times could thus becarried out in quadruplicate in one dish. The test concentrations wereadded aseptically in volumes of 2.5 ml to each compartment for thestandard exposure times, which were 5, 10, 20, 40 and 80 mins. At theend of the individual exposure times, the test solutions were removedfrom the compartments by aspiration. Each set of discs was then washedin situ in two changes of sterile river water (for 5 and 30 mins,respectively) and then incubated in a further 2.5 ml of sterile riverwater, in situ, for 72 h at 16° C. Subsequently, the discs were examinedunder a stereomicroscope, using transmitted dark-ground illumination,and scored for presence or absence of new growth on the agar discsurface. In all cases of doubt, particularly at the borderline betweengrowth and no growth, discs were then transferred to RGY for a further72 h at 16° C. to determine accurately the viability of the myceliumwithin the disc. The test was repeated, substituting malachite greenoxalate for bronopol.

The results were expressed both in terms of effect on zoosporulation andin terms of effect on vegetative growth, and are set out in the tablesbelow:

                  TABLE 1B(i)                                                     ______________________________________                                        Bronopol - Effect on zoosporulation (percentage                               inhibition)                                                                   ______________________________________                                        100%    100%     0       0     0    80    T                                   100%    100%     0       0     0    40    i                                   100%    100%     0       0     0    20    m                                   100%    100%     0       0     0    10    e                                   100%    100%     0       0     0    5     (min)                               1000    100      10      1     0                                              ______________________________________                                         Concentration (mg. 1.sup.-1)                                             

                  TABLE 1B(ii)                                                    ______________________________________                                        Malachite green oxalate - Effect on                                           zoosporulation (percentage inhibition)                                        ______________________________________                                        100%    100%     100%    100%    0    80   T                                  100%    100%     100%    100%    0    40   i                                  100%    100%     100%    100%    0    20   m                                  100%    100%     100%    40-50%  0    10   e                                  100%    100%     100%    40-50%  0    5    (min)                              1000    100      10      1       0                                            ______________________________________                                         Concentration (mg. 1.sup.-1)                                             

                  TABLE 1B(iii)                                                   ______________________________________                                        Bronopol - Effect on vegetative growth (percentage                            inhibition)                                                                   ______________________________________                                        100%     10-20    0       0    0    80    T                                   100%     0        0       0    0    40    i                                   100%     0        0       0    0    20    m                                   10-20%   0        0       0    0    10    e                                   10-20%   0        0       0    0    5     (min)                               1000     100      10      1    0                                              ______________________________________                                         Concentration (mg. 1.sup.-1)                                             

                  TABLE 1B(iv)                                                    ______________________________________                                        Malachite green oxalate - Effect on                                           vegetative growth (percentage inhibition)                                     ______________________________________                                        100%     100%     100%    0    0    80    T                                   100%     100%     100%    0    0    40    i                                   100%     100%     100%    0    0    20    m                                   100%     100%     100%    0    0    10    e                                   100%     100%     100%    0    0    5     (min)                               1000     100%     10      1    0                                              ______________________________________                                         Concentration (mg. 1.sup.-1)                                             

Discussion

Bronopol has a marked effect on immediate zoosporulation with exposuresas little as 5 minutes at concentrations of 100 ppm or (possibly) less.This test uses an agar plug method, in which the agar containing themycelium obviously offers protection to the hyphae, but bronopol stillpenetrates and a 20 minute 1,000 ppm exposure prevents all subsequentgrowth, and this concentration also has a significant effect in as shortas 5 minutes. Furthermore, the lower 100 ppm exposure for 80 minutesalso appears to have some inhibitory effect.

Trial 2

In Vivo Efficacy of Bronopol Against Saprolegnia parasitica Infection inTrout Eggs

Approximately 913,000 rainbow trout eggs were used in this trial, whichwas split between three farm sites. In each case, the eggs werecarefully measured into incubators or trays and very gently immersed inwater at an even temperature, after removing any faeces, blood or white(dead) eggs. The eggs were monitored until they reached the eyed stage,and were then shocked by dropping into water from a height ofapproximately 0.5 m. The purpose of shocking is to kill any weak orinfertile eggs, which turn white due to the rupture of the innermembrane. Dead eggs were removed and counted. The trial was continueduntil the eggs hatched, at which time dead eggs were again removed andcounted. Throughout the trial the degree of fungal infection (attributedto Saprolegnia) was monitored.

The first farm site (Site 1) was run on ground water and the eggs weremaintained in a trough and tray system from stripping to hatching.Incubator volumes were 101 l, with a water exchange rate of 40 l.min⁻¹(the time for water exchange being 2.5 min).

Site 2 was also run on ground water. From stripping to shocking the eggswere kept in vertical incubators, of 20 l volume. The water exchangerate was 13.6 l.min⁻¹ and the time for water exchange 1.5 min. Fromshocking to hatching the eggs were kept in a trough and tray system, theincubator volumes being 145 l and the water exchange rate 27 l.min⁻¹(the water exchange time being 5.4 min). The eggs at Site 2 were dividedinto 3 batches which were tested separately and at different times.

Site 3 was run on river water, and the eggs were maintained in a troughand tray system from stripping to hatching. Incubators were of 190 l involume, with a water exchange rate of 10 l.min⁻¹ and a water exchangetime of 19 min.

At each site (and for each batch at Site 2), the eggs were divided intothree treatment groups, one receiving bronopol treatment, one receivingmalachite green treatment, and one receiving no treatment (to act as anegative control). The number of eggs in each group was as follows:

                  TABLE 2A                                                        ______________________________________                                        Number of eggs per treatment group                                                                 Malachite                                                                              Negative                                        Site   Bronopol      Green    Control                                         ______________________________________                                        1      231,040       115,520  24,320                                          2      108,000       108,000  16 000                                          3      249,382       45,579   24,580                                          ______________________________________                                    

Bronopol at a concentration of 500 g.l⁻¹ was added at the top of theincubators and distributed through the eggs with the incoming water. Thewater flow was then turned off when the treatment was estimated to behalf-way through the trough. A bath lasting 30 minutes was then formedin which the concentration of bronopol was generally in the range of 1mg.l⁻¹ to 50 mg.l⁻¹.

Malachite Green was used as a flush treatment. It was administered atthe top of the incubator and flushed through the eggs with the incomingwater (the water flow was not turned off). This was the usual method onall of the sites. The dosage varied from site to site as follows: Site 115 mg.l⁻¹ ; Site 2 40 mg.l⁻¹ ; Site 3 5 mg.l⁻¹.

Eggs in both bronopol and malachite green groups were treated every dayfrom stripping to hatching.

Fungal growth was assessed visually as the percentage of eggs coveredwith fungal growth, on every second day of the study at Sites 1 and 2,and every day on Site 3. The results are displayed graphically in FIGS.1 to 3 and are summarised in Table 2B below:

                  TABLE 2B                                                        ______________________________________                                        Percentage of Treatment days with degree of fungal growth                     Percentage Malachite            Negative                                      Fungal Growth                                                                            Green       Bronopol Control                                       ______________________________________                                        0%         41.57%      46.63%   28.09%                                        0% to 10%  47.19%      36.52%   35.39%                                        11% to 25% 11.24%      16.85%   8.99%                                         26% to 50% 0.00%       0.00%    16.29%                                        51% to 100%                                                                              0.00%       0.00%    11.24%                                        ______________________________________                                    

Table 2B shows that bronopol has resulted in no fungal growth for 46.63%of the days that the eggs were treated. This can be compared with 41.57%for Malachite Green and 28.09% when no treatment is applied. Bothbronopol and Malachite Green prevented the eggs being covered with morethan 25% fungal growth.

Dead eggs were counted and removed at shocking and at hatching and thenumbers of dead eggs in each case are set out in Tables 2C to 2K below.Tables 2L & 2M show overall mortalities at shocking and hatching,respectively. (Site 1 did not record mortalities at hatching, hence thelower original value in table 2M).

                  TABLE 2C                                                        ______________________________________                                        Site 1 Number of Dead Eggs after Shocking                                                Malachite          Negative                                                   Green    Bronopol  Control                                         ______________________________________                                        Original No. of                                                                            115,520    231,040   24,320                                      Eggs                                                                          No. of Dead Eggs                                                                           12,960     26,201    2,417                                       After Shocking                                                                Percentage of                                                                              11.21%     11.34%    9.93%                                       Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2D                                                        ______________________________________                                        Site 2 (Batch 1) Number of Dead Eggs after Shocking                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            13,800     16,560    5,060                                       Eggs                                                                          No. of Dead Eggs                                                                           650        2,000     223                                         After Shocking                                                                Percentage of                                                                              4.7%       12.1%     4.4%                                        Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2E                                                        ______________________________________                                        Site 2 (Batch 2) Number of Dead Eggs after Shocking                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            43,240     43,320    10,120                                      Eggs                                                                          No. of Dead Eggs                                                                           6,360      8,464     1,700                                       After Shocking                                                                Percentage of                                                                              14.7%      19.5%     16.8%                                       Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2F                                                        ______________________________________                                        Site 2 (Batch 3) Number of Dead Eggs after Shocking                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            57,120     52,020    5,100                                       Eggs                                                                          No. of Dead Eggs                                                                           6,855      3,640     665                                         After Shocking                                                                Percentage of                                                                              12.0%      7.0%      13.0%                                       Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2G                                                        ______________________________________                                        Number of Dead Eggs after Shocking                                                         Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            45,579     249,382   24,580                                      Eggs                                                                          No. of Dead Eggs                                                                           5,331      52,646    4,713                                       After Shocking                                                                Percentage of                                                                              11.70%     21.11%    19.17%                                      Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2H                                                        ______________________________________                                        Site 2 (Batch 1) Number of Dead Eggs after Hatching                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            13,800     16,560    5,060                                       Eggs                                                                          No. of Dead Eggs                                                                           1,000      2,585     477                                         After Shocking                                                                Percentage of                                                                              7.2%       15.6%     9.4%                                        Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2I                                                        ______________________________________                                        Site 2 (Batch 2) Number of Dead Eggs after Hatching                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            43,240     43,320    10,120                                      Eggs                                                                          No. of Dead Eggs                                                                           5,900      9,450     9,600                                       After Shocking                                                                Percentage of                                                                              13.6%      22.3%     94.9%                                       Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2J                                                        ______________________________________                                        Site 2 (Batch 3) Number of Dead Eggs after Hatching                                        Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            57,120     52,020    5,100                                       Eggs                                                                          No. of Dead Eggs                                                                           7,300      4,200     4,100                                       After Shocking                                                                Percentage of                                                                              12.8%      8.1%      80.4%                                       Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2K                                                        ______________________________________                                        Site 3 Number of Dead Eggs after Hatching                                                  Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            45,579     249,382   24,580                                      Eggs                                                                          No. of Dead Eggs                                                                           8,487      47,539    7,203                                       After Shocking                                                                Percentage of                                                                              18.62%     19.06%    36.26%                                      Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2L                                                        ______________________________________                                        Summary of Numbers of Dead Eggs after Shocking                                             Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            275,259    592,322   69,180                                      Eggs                                                                          No. of Dead Eggs                                                                           32,156     92,951    9,718                                       After Shocking                                                                Percentage of                                                                              11.68%     15.70%    14.05%                                      Dead Eggs                                                                     ______________________________________                                    

                  TABLE 2M                                                        ______________________________________                                        Summary of Numbers of Dead Eggs after Hatching                                             Malachite            Negative                                                 Green      Bronopol  Control                                     ______________________________________                                        Original No. of                                                                            159,739    360,282   44,860                                      Eggs                                                                          No. of Dead Eggs                                                                           22,687     63,774    21,380                                      After Shocking                                                                Percentage of                                                                              14.20%     17.70%    47.66%                                      Dead Eggs                                                                     ______________________________________                                    

Conclusion

Bronopol appears to be as effective as Malachite Green at controllingfungal infections in salmonid eggs. The overall mortalities at hatchingfor Malachite Green and bronopol are lower than the negative control,with 14.20%, 17.70% and 47.66% dead eggs, respectively.

Trial 3 In Vivo Efficacy of Bronopol Against Saprolegnia parasiticaInfection in Salmon

Method

105 Atlantic salmon (Salmon salar) of mixed sex aged approximately 8months and weighing approximately 30 g on average where divided intothree treatment groups, each of 35 fish. Each group was kept in freshwater at 12.5° C. in a 210 litre fibreglass tank. The fish were feddaily with 2% body weight of "Biomar" production feed. After two weeks'acclimatisation, all three groups were artificially infected withSaprolegnia parasitica, the date of infection being designated `day 0`of the trial. The groups were then treated, on days 1, 3 and 5, asfollows:

    ______________________________________                                                            Treatment    Treatment                                    Groups   Treatment  concentration                                                                              Duration                                     ______________________________________                                        1        None       --           --                                           2        Malachite  2 mg · 1.sup.-1                                                                   60 min bath                                           green                                                                3        Bronopol   30 mg · 1.sup.-1                                                                  15 min bath                                  ______________________________________                                    

On day 8 of the trial, all fish were culled, and the degree of infectionof each fish assessed according to the following scoring system:

    ______________________________________                                        Degree of fungal infection                                                                         Score                                                    ______________________________________                                        None                 1                                                        Mild (fungus visible on <25%                                                                       2                                                        of fish's surface)                                                            Moderate (fungus visible on 50%                                                                    3                                                        of fish's surface)                                                            Severe (Lesion(s) on fish)                                                                         4                                                        Mortality*           5                                                        ______________________________________                                         (*Dead fish were removed as mortalities occurred, to avoid contamination      of water.)                                                               

Results

The number of fish in each category of the above scoring system, and thetotal score for each group, were as follows:

    ______________________________________                                                                 Malachite                                            SCORE         CONTROL    Green     Bronopol                                   ______________________________________                                        None       1      18         25      23                                       Mild       2      3          1       6                                        Moderate   3      3          1       2                                        Severe     4      3          0       0                                        Mortality  5      8          8       4                                        TOTAL SCORE       85         70      61                                       ______________________________________                                    

The cumulative numbers of mortalities for each day of the trial areillustrated graphically in FIG. 4.

Conclusion

Bronopol proved effective against Saprolegnia infection, and alsoreduced mortalities by 50% compared to both the non-treatment and themalachite green treatment groups.

Trial 4 In Vivo Efficacy of Bronopol Against Ichthyobodo necatrixInfection in Rainbow Trout

Method

Rainbow trout (Oncorhyncus mykiss) having a mean weight of 15 g wereused in this trial, and prior to treatment were confined in a singlepond with 6 individuals heavily infected with Ichthyobodo. Infection ofthe previously healthy individuals was confirmed after 5 days bymicroscopy of mounted gill specimens. Bronopol treatment wasadministered to a group of 20 fish using the same method as in Trial 3.The Trial was repeated using a group of 19 infected fish treated withformalin (a standard treatment for Ichthyobodo), with a further group of20 infected fish receiving no treatment and acting as a control. At 7days after treatment the mean number of parasites per gill filament wasassessed in each group (by counting the number of parasites on 10filaments of each of 5 fish), and at 14 days a further count wasconducted, with individual fish being either categorised as having noinfection, or being placed in one of four infected categories, accordingto the degree of infection.

Results

    ______________________________________                                                                     GROUP                                            Days Post                                                                            Infection             No treatment                                     Treatment                                                                            Level       Bronopol  Control  Formalin                                ______________________________________                                        7      mean numbers                                                                              <0.5      2.0      0                                              of parasites                                                                  per gill                                                                      filament                                                               14     percentage in                                                                 each category:                                                                -           50        0        100                                            +           43        29       0                                              ++          7         29       0                                              +++         0         18       0                                              ++++        0         23       0                                       ______________________________________                                    

No Mortalities Occurred

Conclusion

Bronopol was shown to have good activity against Ichthyobodo infectionin rainbow trout. Although less effective than formalin, its use carriesless risk. It may be inferred that bronopol will have similar efficacyagainst Ichthyobodo infection in other salmonids, and in fish andaquatic organisms in general, and will also be useful in the prophylaxisof the disease.

Trial 5 In Vitro Efficacy of Bronopol Against Cytophaga psychrophila

Method

First, the Minimal Bactericidal Concentration (MBC) for bronopol againstCytophaga psychrophila was established by growing the organism in broth,and then adding different dilutions of bronopol to establish whatconcentration would kill the Cytophaga.

Then based on the MBC results, the contact times required to kill theCytophaga were investigated. A known number of viable bacteria wereadded to distilled water and exposed to various concentrations ofbronopol for 2-40 minutes. Then the bacteria were extracted byfiltration, and the extracted bacteria tested for viability byinvestigating their growth in culture medium by measuring opticaldensity at 520 nm.

Results

The results are set out graphically in FIG. 5. The minimum testconcentration which was shown to be effective was 5 mg.l⁻¹ (ppm), but itis likely that the actual minimum effective concentration lies between 1mg.l⁻¹ and 5 mg.l⁻¹. It was also found that an exposure time of up to 40mins was required to prevent bacterial growth after re-inoculation ontofresh media when concentrations of between 5 mg.l⁻¹ and 400 mg.l⁻¹ wereused, with the necessary exposure time being reduced to around 4 minutesat a concentration of 1000 mg.l⁻¹ and around 2 minutes at 2000 mg.l⁻¹.

Conclusion

Bronopol was shown to be active against Cytophaga psychrophila atconcentrations as low as 5 mg.l⁻¹. Cytophaga is an example of the groupof bacteria known as myxobacteria, which are characterised by aprotective muco-polysaccharide layer, and which are generally resistantto disinfectants (for example, the dose of iodophor disinfectantrequired to kill Cytophaga is approximately 2000 mg.l⁻¹). Based on theknown uses of bronopol, this activity is therefore surprising, and mayindicate activity against myxobacteria in general. Bronopol is likely toprove a suitable disinfectant treatment for fish tanks and equipment,and may also be of use in the treatment and/or prophylaxis for thevarious diseases caused by this organism in trout and other salmonids,and also (when these occur) in other fish and aquatic organisms.

Trial 6 In Vivo Efficacy of Bronopol Against Flavobacteriumbranchophilum Infection in Rainbow Trout

Seven tanks of 150 rainbow trout each, at a stocking density of 100g.l⁻¹, were included in this experiment. The fish averaged 15 g in size.Water temperature was 11° C. and turned over at a rate of once per hour.

One tank of fish was maintained as a negative control and was neitherchallenged or treated. An additional tank was challenged, but nottreated, to act as a positive control. Two tanks were treated withchloramine-T (a conventional treatment) at a concentration of 10 mgl⁻¹.Three tanks were treated with bronopol, one each at the followingconcentrations: 5, 25 and 50 mg.l⁻¹.

Fish were challenged on day 0, and tanks were treated with eitherchloramine-T or bronopol on days 2 and 4 of the experiment. All sixtanks which were experimentally infected with F. branchiophilum weretreated with the respective therapeutants simultaneously for one hour ina static bath with aeration.

The efficacy of each treatment was evaluated by clinical signs, gillbacterial antigen levels measured by enzyme immunoassay (EIA), mortalityrates and cumulative mortality and finally by histological examination.

Gill samples for EIA were collected from five fish in each tank beforeexperimental infection, and before treatment. Further gill samples werecollected 6 and 36 hours after the initial treatment, after which thegroups were retreated with identical concentrations of the sametherapeutant, in an identical manner. Gill samples from five fish fromeach tank were again collected 6 and 36 hours after the secondtreatment. Mortality in each tank was recorded each day. The results arepresented graphically in FIGS. 6 to 8.

Based on the mortality and EIA data, the lowest concentration ofbronopol (5 mg.l⁻¹, FIG. 5) was found to be ineffective. Mortality wasinversely related to the dose of bronopol (FIG. 6).

It is evident that mortality corresponds closely with the bacterialantigen levels detected on the gills, and that the EIA is a useful toolfor monitoring the efficacy of a therapeutant's ability to eliminatebacteria from the gill surface. As with mortality, the bacterial gillantigen concentration at most sample times, is inversely proportional tothe dose of therapeutant (FIG. 8). The two highest concentrations ofbronopol effectively eliminated the bacteria from the gill surface afterthe second treatment (FIG. 8). It is clear that bronopol is effectiveagainst Flavobacterium branchophilum, and that in the trial bronopol at25 mg.l⁻¹ and 50 mg.l⁻¹ reduced mortalities among the infected fish,both compared to the untreated fish, and to the fish treated withchloramine-T.

Trial 7 In Vivo Efficacy of Bronopol Against Saprolegnia parasitica inBrown Trout

Method

This trial set out to assess the efficacy of bronopol in the treatmentof brown trout naturally infected with Saprolegnia parasitica. 40 fishwere used in the trial, and these were divided equally into two groupsheld in separate tanks. A moribund fish heavily infected withSaprolegnia parasitca was placed in each tank, with the intention ofinfecting the other fish naturally. On days 1, 3 and 5 of the trial thefish in one group were treated to a 50 mg.l⁻¹ bath of bronopol for 15min, while the fish in the second group were left untreated and acted asa control. The degree of infection of the fish in both groups wasassessed on day 7 of the trial.

Results

In both groups the moribund fish died on day 1 of the trial, but in eachcase was left in the tank to ensure a good challenge to the remainingfish. On day 7 of the trial all of the fish in the control group werenoted to be severely affected by Saprolegnia, and were killed onhumanitarian grounds. In contrast, the fish in the bronopol treatmentgroup were all healthy and had no visual signs of Saprolegnia infection.

Conclusion

Repeated treatment with bronopol at a concentration of 50 mg.l⁻¹ appearsto be highly effective as a treatment for Saprolegnia infection in browntrout, and/or as a prophylactic to prevent infection.

What is claimed is:
 1. A method of treatment of an aquatic organismsuffering from a disease selected from the group consisting of fungalinfections, flagellate protozoan infections, ciliate protozoaninfections, bacterial gill disease, and myxobacterial infections, themethod comprising the step of administering to said organism, as atopical treatment, a pharmaceutically effective amount of a solutioncontaining, as the sole or principal active ingredient against saiddisease, a substance selected from the group consisting of bronopol(2-bromo-nitropropane-1,3-diol) and substances which release bronopolwhen placed in an aquatic environment.
 2. A method as claimed in claim1, for the treatment of a disease caused by a causative organismselected from the group consisting of: Saprolegnia parasitica,Ichthyobodo necatrix, Icthyophthirius multifiliis, Flavobacteriumbranchiophilum and Cytophaga psychrophila.
 3. The method of claim 2, forthe treatment of an organism selected from the group consisting ofsalmonid fish and salmonid fish eggs.
 4. The method of claim 3, whereinthe salmonid fish is selected from the group consisting of trout andsalmon.
 5. The method of claim 1, wherein the concentration of bronopolin the treatment bath is in the range 1 to 1,000 mg.l⁻¹ (ppm).
 6. Themethod of claim 5, wherein the concentration of bronopol in thetreatment bath is in the range 5 to 200 mg.l⁻¹ (ppm).
 7. The method ofclaim 6, wherein the concentration of bronopol in the treatment bath isin the range 10 to 100 mg.l⁻¹ (ppm).
 8. A method of disinfectingequipment selected from fish tanks and equipment for use in thehusbandry of fish, the method comprising the step of exposing saidequipment to a solution containing as the sole or principal disinfectingagent a substance selected from the group consisting of bronopol(2-bromo-2-nitropropane-1,3-diol) and substances which release bronopolwhen placed in aquatic environment.
 9. The method of claim 8, whereinthe concentration of bronopol is in the range 1 to 2,000 mg.l⁻¹ (ppm).10. The method of claim 9, wherein the concentration of bronopol is inthe range 5 to 1,000 mg.l⁻¹ (ppm).
 11. The method of claim 8, whereinsaid equipment is exposed to said solution for a period of time in therange 2 to 40 minutes.
 12. A treatment bath for the treatment of anaquatic organism suffering from a disease selected from the groupconsisting of: fungal infections, flagellate protozoan infections,ciliate protozoan infections, bacterial gill disease, and myxobacterialinfections, the treatment bath containing as the sole or principalactive ingredient against said disease a substance selected from thegroup consisting of: bronopol and substances which release bronopol whenplaced in an aquatic environment, at a concentration in the range 1 to1,000 mg.l⁻¹ (ppm).
 13. A treatment bath according to claim 12, whereinthe concentration of bronopol is in the range 5 to 200 mg.l⁻¹ (ppm). 14.A treatment bath according to claim 13, wherein the concentration ofbronopol is in the range 10 to 100 mg.l⁻¹ (ppm).